The systematic characterization of gene function, and the understanding of how gene dysfunction leads to disease states, is one of the largest challenges in biology today. The number of genes multiplied by the number of cellular states in which they are expressed emphasizes the need for scalable, comprehensive, unbiased, and robust techniques for perturbing genes to ascertain their functions. CRISPR-Cas9 technology represents the most promising tool yet described for experimental manipulation of the genome, and a central focus of Core C is the development, pressure testing, and deployment at-scale of this technology. Core C will support Research Projects 1 and 2 in this U19 proposal, ?Defining regulators of immunity to acute infection using CRISPR screens.? Specifically, we will work with the computational core (Core B) and the individual investigators in each of the two Research Projects to curate a set of target genes for screening in mouse models (Aim 1). We will generate pooled libraries of gRNA for each project using optimized design algorithms that maximize the efficiency of the reagents, enabling the use of small numbers of gRNAs per gene and thus the screening of more genes. These libraries will then be produced as lentivirus for delivery into cells. We will then work with the mouse screening core (Core D) to determine optimal screening conditions for each of the individual projects (Aim 2). Core D will execute the in vivo screens and provide genomic DNA samples to Core C for processing and analysis. Core C will PCR the gRNA inserts, sequence the product via Illumina, and use customized software to deconvolute the sequencing reads. Finally, Core C will combine information from multiple gRNAs targeting each gene to define a hit list for each screen, using the STARS algorithm. This hit list is the starting point for systematic network analysis by Core B. Following primary screens, Core C will support the customized creation of individual reagents necessary for focused follow-up (Aim 3). This support includes the design and production of individual gRNA reagents, creation of customized vectors, and implementation of alternative perturbational technologies, such as CRISPRa, CRISPRi, ORFs, and RNAi. Together, these three aims will accelerate the research of both Research Projects and enable deep functional knowledge across multiple models of immunity.